U.S. patent application number 15/740981 was filed with the patent office on 2018-07-05 for immune checkpoint chimeric antigen receptors therapy.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Ivan M. Borrello, Susan Lee, Kimberly A. Noonan, Drew M. Pardoll.
Application Number | 20180185434 15/740981 |
Document ID | / |
Family ID | 57609024 |
Filed Date | 2018-07-05 |
United States Patent
Application |
20180185434 |
Kind Code |
A1 |
Borrello; Ivan M. ; et
al. |
July 5, 2018 |
IMMUNE CHECKPOINT CHIMERIC ANTIGEN RECEPTORS THERAPY
Abstract
In some aspects, the embodiments relate to compositions and
methods related to chimeric transmembrane proteins. The chimeric
transmembrane proteins may comprise the extracellular domain of an
inhibitory receptor, and an intracellular signaling domain that can
activate an immune response.
Inventors: |
Borrello; Ivan M.;
(Baltimore, MD) ; Lee; Susan; (Baltimore, MD)
; Noonan; Kimberly A.; (Baltimore, MD) ; Pardoll;
Drew M.; (Brookeville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Family ID: |
57609024 |
Appl. No.: |
15/740981 |
Filed: |
June 29, 2016 |
PCT Filed: |
June 29, 2016 |
PCT NO: |
PCT/US16/40010 |
371 Date: |
December 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62186108 |
Jun 29, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70521 20130101;
C07K 19/00 20130101; A61P 35/02 20180101; C07K 14/705 20130101;
A61K 48/005 20130101; C12N 15/8201 20130101; C07K 14/7051 20130101;
A61K 35/17 20130101; A61K 38/17 20130101; C07K 14/70596 20130101;
C12N 15/62 20130101; A61K 38/1709 20130101; A61K 38/005 20130101;
A61K 38/00 20130101 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61K 38/17 20060101 A61K038/17; C07K 14/725 20060101
C07K014/725; C07K 14/705 20060101 C07K014/705; C07K 19/00 20060101
C07K019/00; C12N 15/62 20060101 C12N015/62; A61K 35/17 20060101
A61K035/17; A61K 48/00 20060101 A61K048/00; A61P 35/02 20060101
A61P035/02; C12N 15/82 20060101 C12N015/82 |
Claims
1. A chimeric transmembrane protein, comprising: the extracellular
domain of an inhibitory receptor; and an intracellular signaling
domain that can activate an immune response, wherein the
intracellular signaling domain comprises a portion of an
intracellular signaling protein.
2. The protein of claim 1, wherein the protein comprises a sequence
selected from the group consisting of SEQ ID NOs: 7-10.
3-5. (canceled)
6. The protein of claim 1, wherein the intracellular signaling
domain comprises kinase activity.
7. The protein of claim 1, wherein the intracellular signaling
domain comprises a phosphorylation site.
8. The protein of claim 1, wherein the protein comprises the
transmembrane domain of the inhibitory receptor or the
transmembrane domain of the intracellular signaling protein.
9. (canceled)
10. The protein of claim 1, wherein the inhibitory receptor reduces
immune activity upon binding a native agonist.
11. The protein of claim 1, wherein the inhibitory receptor can
reduce T cell proliferation, T cell survival, cytokine secretion,
or immune cytolytic activity upon binding a native agonist.
12. The protein of claim 1, wherein the inhibitory receptor is a
lymphocyte inhibitory receptor.
13. The protein of claim 12, wherein the inhibitory receptor is
CTLA-4, PD-1, LAG-3, or Tim-3.
14. (canceled)
15. (canceled)
16. The protein of claim 1, wherein the intracellular signaling
protein increases immune activity.
17. The protein of claim 1, wherein the intracellular signaling
protein can enhance T cell proliferation, T cell survival, cytokine
secretion, or immune cytolytic activity.
18. The protein of claim 1, wherein the intracellular signaling
protein is a transmembrane protein or the intracellular signaling
protein can bind a native transmembrane protein.
19. The protein of claim 1, wherein the intracellular signaling
protein is a lymphocyte protein.
20. The protein of claim 19, wherein the intracellular signaling
protein is CD3.zeta., 4-1BB, or CD28.
21. (canceled)
22. The protein of claim 1, further comprising a suicide
domain.
23. The protein of claim 22, wherein the suicide domain has
thymidine kinase activity or the suicide domain is a caspase.
24. (canceled)
25. A nucleic acid encoding the chimeric transmembrane protein of
claim 1.
26. (canceled)
27. A recombinant cell, comprising the chimeric transmembrane
protein of claim 1.
28. The cell of 27, wherein the cell is a T cell, a tumor
infiltrating lymphocyte ("TIL"), a marrow infiltrating lymphocyte
("MIL"), or a lymphocyte.
29-39. (canceled)
40. A method for increasing an immune response or treating a
neoplasm in a subject, comprising administering to the subject the
recombinant cell of claim 27.
41-54. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/186,108, filed Jun. 29, 2015, which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0002] The large majority of patients with malignancies will die
from their disease. One approach to treating these patients is to
genetically modify T cells to target antigens expressed on tumor
cells through the expression of chimeric antigen receptors (CARs).
CARs are antigen receptors that are designed to recognize cell
surface antigens in a human leukocyte antigen-independent manner.
Outside of the successes with CD19-targeted approaches, attempts at
using genetically modified cells expressing CARs to treat other
malignancies have met with limited success.
[0003] Recently, checkpoint inhibiting antibodies targeting CTLA-4
(ipilimumab) and PD-1 (nivolumab, pembrolizumab) have shown
considerable activity in the treatment of various malignancies
including metastatic melanoma, non small cell lung cancer (NSCLC)
and Hodgkin's lymphoma. These data demonstrate how checkpoint
blockade represents a major obstacle to effective immunotherapy by
overcoming T cell anergy.
SUMMARY
[0004] In some aspects, the embodiments relate to a chimeric
transmembrane protein, comprising the extracellular domain of an
inhibitory receptor and an intracellular signaling domain that can
activate an immune response. The extracellular domain may be, for
example, an extracellular domain from CTLA-4, PD-1, LAG-3, or
Tim-3. The intracellular signaling domain may be, for example, the
intracellular signaling domain of CD3.zeta., 4-1BB, or CD28. In
some aspects, the embodiments relate to a nucleic acid encoding a
chimeric transmembrane protein as described herein.
[0005] In some aspects, the embodiments relate to cells, comprising
a nucleic acid encoding a chimeric transmembrane protein as
described herein. In some aspects, the embodiments relates to
cells, comprising a chimeric transmembrane protein as described
herein.
[0006] In some aspects, the embodiments relate to methods for
making recombinant cells, comprising transfecting cells with a
nucleic acid encoding a chimeric transmembrane protein as described
herein.
[0007] In some aspects, the embodiments relate to methods for
increasing an immune response in a subject, comprising
administering to the subject a recombinant cell as described
herein. In some aspects, the embodiments relate to methods for
treating a neoplasm in a subject, comprising administering to the
subject a recombinant cell as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) encoding a
chimeric transmembrane protein, comprising a leader peptide from
CD8 ("CD8a LP"), the extracellular domain of mouse PD-1 ("PD-1
ECD"), and the transmembrane and intracellular domains of mouse
4-1BB ("4-1BB TM" and "4-1BB ICD", respectively). The reverse
complement of the nucleotide sequence (SEQ ID NO:2) is also shown.
Codons were optimized for expression in mouse lymphocytes.
[0009] FIG. 2 shows a nucleotide sequence (SEQ ID NO:3) encoding a
chimeric transmembrane protein, comprising a leader peptide from
CD8 ("CD8a LP"), the extracellular domain of human PD-1 ("PD-1
ECD"), and the transmembrane and intracellular domains of human
4-1BB ("4-1BB TM" and "4-1BB ICD", respectively). The reverse
complement of the nucleotide sequence (SEQ ID NO:4) is also shown.
Codons were optimized for expression in human lymphocytes.
[0010] FIG. 3 shows flow cytometry results for Lenti-X 293T cells
transfected with a mCherry gene and a nucleic acid encoding a
chimeric transmembrane protein (SEQ ID NO:1), comprising the
extracellular domain of PD-1 using the transfection protocol
described in Example 2, infra. FIG. 3 shows that the nucleic acid
is expressed in 293T cells.
[0011] FIG. 4 shows flow cytometry results for Lenti-X 293T cells
transduced with a mCherry gene and a nucleic acid encoding a
chimeric transmembrane protein (SEQ ID NO:1), comprising the
extracellular domain of PD-1 and the intracellular domain of 4-1BB,
using the transduction protocol described in Example 1, infra.
Cells were transduced in 1 well of a 6-well plate with 1.9 mL of
virus. FIG. 4 shows that the nucleic acid is expressed in 293T
cells.
[0012] FIG. 5 shows flow cytometry results for Lenti-X 293T cells
transduced with a mCherry gene and a nucleic acid encoding a
chimeric transmembrane protein (SEQ ID NO:1), comprising the
extracellular domain of PD-1 and the intracellular domain of 4-1BB,
using the transduction protocol described in Example 1, infra.
Cells were transduced in 1 well of a 6-well plate with 0.38 mL of
virus. FIG. 5 shows that the nucleic acid is expressed in 293T
cells.
[0013] FIG. 6, Panel A and Panel B illustrates that MILs comprising
a chimeric receptor having a PD-1 extracellular domain, a 4-1BB
transmembrane domain, and a 4-1BB intracellular domain do not
negatively affect tumor specificity.
DETAILED DESCRIPTION
[0014] CAR therapy has shown significant promise to date. CD19 CARs
targeting chronic lymphocytic leukemia (CLL) and more recently,
acute lymphoblastic leukemia (ALL) have met with notable success.
Interestingly, CARs targeting other antigens have not provided
similar clinical responses. One limitation of such antigen-targeted
approaches is their therapeutic applicability, which is limited
only to the diseases expressing particular surface receptors and
the limitations of targeting a single tumor antigen that have
resulted in relapses with antigen-loss variants.
[0015] A major hurdle in tumor immunology is the induction of
tumor-specific tolerance which limits the intrinsic anti-tumor
efficacy of many cell based approaches. Recent studies have shown
significant clinical efficacy by targeting checkpoint inhibitors
leading to the approval of anti-CTLA-4 and anti-PD-1 for metastatic
melanoma. In some aspects, the embodiments relate to a chimeric
receptor, comprising an extracellular domain expressing of a
checkpoint inhibitor and an activating intracellular domain. This
has the advantage of hijacking the tolerogenic mechanisms into
activating signals. This approach can be used in all clinical
situations in which T cell anergy is a major aspect of the
pathogenesis of the disease and where the antigen specificity is
provided by the endogenous T cell repertoire.
[0016] In some aspects, the embodiments relate to a chimeric
transmembrane protein, comprising an extracellular domain of an
inhibitory receptor, a transmembrane domain, and an intracellular
signaling domain. In some embodiments, the intracellular signaling
domain can activate an immune response. The intracellular signaling
domain may comprise a portion of an intracellular signaling
protein. In some embodiments, the intracellular domain can be used
to maintain the activation of a cell, such as a T-cell.
[0017] In some embodiments, the extracellular domain can transduce
a signal to the intracellular signaling domain. For example, the
extracellular domain may transduce a signal to the intracellular
signaling domain upon binding an agonist of the native inhibitory
receptor.
[0018] Signal transduction may comprise oligomerization of the
protein. Oligomerization may comprise homo-oligomerization or
hetero-oligomerization. Oligomerization may comprise dimerization
of the protein, i.e., homo-dimerization with a second chimeric
transmembrane protein or hetero-dimerization with a different
protein.
[0019] Signal transduction may comprise phosphorylation. For
example, the intracellular signaling domain may comprise kinase
activity and/or a phosphorylation site. Signal transduction may
comprise autophosphorylation, e.g., autophosphorylation of the
intracellular signaling domain.
[0020] In some embodiments, the protein comprises a transmembrane
domain. In some embodiments, the protein is an integral membrane
protein. For example, the protein may be a type 1 membrane protein,
a type 2 membrane protein, or a multi-spanning membrane protein. In
some embodiments, the protein comprises the transmembrane domain of
the inhibitory receptor. In some embodiments, the protein comprises
the transmembrane domain of the intracellular signaling protein.
The chimeric transmembrane protein may comprise a signal peptide,
e.g., to translocate the extracellular domain across a cell
membrane. In some embodiments, the transmembrane domain comprises
the sequence of IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 5). In some
embodiments, the chimeric transmembrane protein comprises a signal
peptide derived from CD8. In some embodiments, the signal peptide
comprises the CD8 leader peptide. In some embodiments, the signal
peptide comprises MALPVTALLLPLALLLHAARP (SEQ ID NO: 6).
[0021] In some embodiments, the extracellular domain is the
extracellular domain of an inhibitory receptor. In some
embodiments, the extracellular domain comprises a ligand-binding
domain, e.g., the agonist-binding domain of the inhibitory
receptor. In some embodiments, the extracellular domain comprises
sufficient structure to transduce a signal across the membrane in
response to ligand binding. Without being bound to any particular
theory, for inhibitory receptors that transduce a signal by
oligomerization mediated by a multivalent ligand, the mere presence
of a ligand-binding domain may be sufficient structure to transduce
a signal across the membrane in response to ligand binding. Without
being bound to any particular theory, for inhibitory receptors that
transduce a signal by altering the orientation of a transmembrane
domain relative to the cell membrane, the extracellular domain may
require native structure between the ligand-binding domain and
transmembrane domain to transduce a signal across the membrane in
response to ligand binding. For example, an extracellular domain
may comprise the native sequence of the inhibitory receptor from
its ligand-binding domain to its transmembrane domain.
[0022] The native inhibitory receptor can be a human inhibitory
receptor or a mouse inhibitory receptor. Thus, the extracellular
domain may comprise a human or mouse amino acid sequence. In some
embodiments, the origin of the native inhibitory receptor is
selected to match the species of a subject that is being treated,
e.g., to avoid an immune response against the chimeric
transmembrane protein. Nevertheless, the native inhibitory receptor
may be selected from a different species, e.g., for convenience.
Accordingly, the chimeric protein may or may not be
xenogeneic-derived relative either to the species of cell in which
the protein is expressed or the subject to which the protein is
administered.
[0023] In some embodiments, the native inhibitory receptor is
selected from proteins that reduce immune activity upon binding a
native agonist. For example, the native inhibitory receptor may
reduce T cell proliferation, T cell survival, cytokine secretion,
or immune cytolytic activity upon binding a native agonist. The
native inhibitory receptor may be a lymphocyte inhibitory receptor
(i.e., the inhibitory receptor may be expressed on lymphocytes,
such as T cells). For example, the native inhibitory receptor may
be expressed on T cells, and the binding of an agonist to the
native inhibitory receptor may cause cell signaling that disfavors
T cell proliferation, T cell survival, cytokine secretion, or
immune cytolytic activity.
[0024] In some embodiments, the native inhibitory receptor may be
CTLA-4 (cytotoxic T-lymphocyte-associated protein 4; CD152), PD-1
(Programmed cell death protein 1; CD279), LAG-3
(Lymphocyte-activation gene 3; CD223), or Tim-3 (T cell
immunoglobulin mucin-3). Thus, in some embodiments, the
extracellular domain may be the extracellular domain from CTLA-4,
PD-1, LAG-3, or Tim-3. The inhibitory receptor may be PD-1. In some
embodiments, the transmembrane protein comprises the extracellular
domain of PD-1. In some embodiments, the sequence of the
extracellular domain comprises
TABLE-US-00001 (SEQ ID NO: 7)
PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRM
SPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT
YLCGAISLAPKAQIKESLRAELRVTERRAEVPTARPSPSPRPAGQFQTL V..
[0025] In some embodiments, the intracellular signaling domain is
the signaling domain of an intracellular signaling protein. In some
embodiments, the intracellular signaling domain may comprise kinase
activity or a phosphorylation site. The intracellular signaling
domain can, in some embodiments, activate a signaling molecule,
such as a kinase or phosphorylase, e.g., following signal
transduction across a cell membrane. The intracellular signaling
domain may signal through a downstream kinase or a
phosphorylase.
[0026] The intracellular signaling protein may be a human protein
or a mouse protein. Thus, the intracellular signaling domain may
comprise a human or mouse amino acid sequence. In some embodiments,
the intracellular signaling protein is selected to match the
species of a subject and cell that is being used for treatment,
e.g., so that the signaling domain may utilize the cell's cytosolic
machinery to activate downstream signaling molecules. Nevertheless,
the intracellular signaling protein may be selected from a
different species, e.g., for convenience, such as described
above.
[0027] In some embodiments, the intracellular signaling protein
increases immune activity. Thus, signal transduction via the
chimeric transmembrane protein can result in a signal cascade that
increases immune activity, wherein the intracellular signaling
domain mediates the intracellular signaling cascade. In some
embodiments, the intracellular signaling protein can enhance T cell
proliferation, T cell survival, cytokine secretion, or immune
cytolytic activity. In some embodiments, the intracellular
signaling protein is a transmembrane protein or the intracellular
signaling protein can bind a native transmembrane protein. The
intracellular signaling protein may be a lymphocyte protein (i.e.,
the intracellular signaling protein may be expressed on
lymphocytes, such as T cells). In some embodiments, the
intracellular signaling protein is CD3.zeta. (T-cell surface
glycoprotein CD3 zeta chain; CD247), 4-1BB (tumor necrosis factor
receptor superfamily member 9; CD137), or CD28 (T-cell-specific
surface glycoprotein CD28; Tp44). Thus, the intracellular signaling
protein may comprise a signaling domain from CD3.zeta., 4-1BB, or
CD28. The intracellular signaling protein may be 4-1BB. Thus, the
intracellular signaling protein may comprise a signaling domain
from 4-1BB. In some embodiments, the intracellular domain
comprises
TABLE-US-00002 (SEQ ID NO: 8)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
[0028] In some embodiments, the chimeric transmembrane protein
comprises a suicide domain, i.e., to kill a recombinant cell
comprising the protein. The suicide domain may comprise thymidine
kinase activity or caspase activity. For example, the suicide
domain may be a thymidine kinase or a caspase. In some embodiments,
the suicide domain is the thymidine kinase domain of HSV thymidine
kinase ("HSV-TK") or the suicide domain comprises a portion of
caspase 9.
[0029] In some aspects, the embodiments relates to a nucleic acid
molecule encoding a chimeric transmembrane protein as described
herein. The nucleic acid molecule may comprise a promoter, wherein
the promoter is operably linked to a nucleotide sequence encoding
the chimeric transmembrane protein, e.g., for expression of a
chimeric transmembrane protein in a recombinant cell. In some
embodiments, the promoter is a constitutive promoter. In some
embodiments, the promoter is a cell specific promoter. In some
embodiments, the promoter is a tissue specific promoter.
[0030] The nucleic acid molecule may comprise the sequence set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The
nucleic acid molecule may comprise at least about 100, 200, 300,
400, 500, 600, or 700 consecutive nucleotides in the sequence set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The
nucleic acid molecule may comprise a nucleotide sequence having at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence homology with the nucleotide sequence set forth in SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The nucleic acid
molecule may comprise a nucleotide sequence having at least about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
homology with at least about 100, 200, 300, 400, 500, 600, or 700
consecutive nucleotides in the nucleotide sequence set forth in SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. For example, the
nucleic acid molecule may comprise a nucleotide sequence having at
least 95% sequence homology with at least 100 consecutive
nucleotides in the nucleotide sequence set forth in SEQ ID
NO:3.
[0031] In some embodiments, the nucleic acid molecule encodes an
amino acid sequence as described herein and/or in the drawings. In
some embodiments, the nucleic acid molecule encodes an amino acid
sequence comprising one or more amino acid sequences set forth in
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the
nucleic acid molecule may comprise a nucleotide sequence that
encodes an amino acid sequence having at least about 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with a
nucleotide sequence set forth herein and/or in the drawings.
Homology can be identity or similarly in the context of a protein.
Sequence homology may refer to sequence identity in the context of
a nucleic acid molecule. Homology can be used by employing routine
tools such as Expasy, BLASTp, Clustal, and the like using default
settings.
[0032] In some embodiments, the chimeric transmembrane protein
comprises one or more amino acid sequences set forth in the
following table:
TABLE-US-00003 Sequence SEQ ID NO IISFFLALTSTALLFLLFFLTLRFSVV 5
MALPVTALLLPLALLLHAARP 6 PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 7
SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ
PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY
LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLV
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE 8 EEEGGCEL
IISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLY 9
IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTC 10
SFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQ
PGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY
LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP
SPSPRPAGQFQTLVIISFFLALTSTALLFLLFFLTLR
FSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCEL
MALPVTALLLPLALLLHAARPPGWFLDSPDRPW 11
NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNW
YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLP
NGRDFHMSVVRARRNDSGTYLCGAISLAPKAQI
KESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTL
VIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLL GCSCRFPEEEEGGCEL
[0033] In some embodiments, the chimeric transmembrane protein
comprises an amino acid sequence having at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with
one of the amino acid sequences set forth herein.
[0034] Variants of the amino acid sequences described herein may be
included in various embodiments. The term "variant" refers to a
protein or polypeptide in which one or more (e.g., 1, 2, 3, 4,
etc.) amino acid substitutions, deletions, and/or insertions are
present as compared to the amino acid sequence of a protein or
polypeptide, and the term includes naturally occurring allelic
variants and alternative splice variants of a protein or
polypeptide. The term "variant" includes the replacement of one or
more amino acids in an amino acid sequence with a similar or
homologous amino acid(s) or a dissimilar amino acid(s). Some
variants include alanine substitutions at one or more amino acid
positions in an amino acid sequence. Other substitutions include
conservative substitutions that have little or no effect on the
overall net charge, polarity, or hydrophobicity of the protein.
Conservative substitutions may have insignificant effect on the
function of the chimeric transmembrane protein. In some
embodiments, the function can be the specificity of a protein when
expressed in a lymphocyte, e.g., a marrow-infiltrating lymphocyte
(MIL), such as described in Example 3. One of skill in the art can
determine if a substitution affects the function of a chimeric
transmembrane protein by comparing to the sequences provided herein
using a protocol identical to, or analogous to, Example 3.
Non-limiting exemplary conservative substitutions are set forth in
the table below. According to some embodiments, a chimeric
transmembrane protein has at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity with an amino acid sequence
described herein.
Conservative Amino Acid Substitutions
TABLE-US-00004 [0035] Basic: arginine lysine histidine Acidic:
glutamic acid aspartic acid Uncharged Polar: glutamine asparagine
serine threonine tyrosine Non-Polar: phenylalanine tryptophan
cysteine glycine alanine valine proline methionine leucine
isoleucine
The table below sets out another scheme of conservative amino acid
substitutions.
TABLE-US-00005 Original Residue Conservative Substitutions Ala Gly;
Ser; Thr Arg Lys; Gln Asn Gln; His; Ser Asp Glu; Asn Cys Ser Gln
Asn; Ser; Asp; Glu Glu Asp; Gln; Lys Gly Ala; Pro; Asn His Asn;
Gln; Tyr Ile Leu; Val; Met; Val; Phe Leu Ile; Val; Met; Phe Lys
Arg; Gln Met Leu; Tyr; Ile; Val; Phe Pro Ser; Thr; Ala; Gly Phe
Met; Leu; Tyr; Trp Ser Thr; Gly; Asn; Asp Thr Ser; Asn Trp Tyr; Phe
Tyr Trp; Phe Val Ile; Leu; Met; Phe
Accordingly, in some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
of the amino acid residues of an amino acid sequence disclosed
herein are modified with conservative substitutions. In some
embodiments, only 1, 2, 3, 4 or 5 amino acid residues are
substituted with conservative substitutions.
[0036] In some embodiments, the chimeric transmembrane protein
comprises a sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or a variant
thereof. SEQ ID NO: 10 is a combination of SEQ ID NO: 5, 7, and 8.
SEQ ID NO: 11 is a combination of SEQ ID NO: 5, 6, 7, and 8. In
some embodiments, the sequence of SEQ ID NO: 6 is replaced with
another signal peptide or leader sequence, that can assist in
trafficking the chimeric transmembrane protein to the extracellular
membrane. In some embodiments, the transmembrane domain, e.g., SEQ
ID NO: 5, is replaced with a different transmembrane protein. In
some embodiments, the transmembrane domain is the transmembrane
domain of PD-1. In some embodiments, the transmembrane domain is
the transmembrane domain of 4-1BB.
[0037] In some aspects, the embodiments relate to a recombinant
cell, comprising a nucleic acid as disclosed herein. In some
embodiments, the embodiments relate to a recombinant cell,
comprising a chimeric transmembrane protein as described herein. In
some embodiments, the cell comprises a chimeric protein comprising
a protein of SEQ ID NO: 5, 6, 7, 8, 9, 10, or 11 or a variant
thereof. In some embodiments, the cell is a lymphocyte. The cell
may be a T cell. In some embodiments, the cell may be a
tumor-infiltrating lymphocyte ("TIL") or a marrow infiltrating
lymphocyte ("MIL").
[0038] In some embodiments, the cell comprising a chimeric
transmembrane protein described herein persist longer and/or remain
in an active state longer in a subject when administered to the
subject as compared to a cell without a chimeric transmembrane
protein.
[0039] In some aspects, the embodiments relate to a method for
making a recombinant cell, comprising transfecting a cell with a
nucleic acid molecule as described herein. In some aspects, the
embodiments relate to a method for making a recombinant cell,
comprising transfecting a cell with a nucleic acid molecule
encoding an amino acid sequence as described herein. The nucleic
acid molecule may be a plasmid. The cell can be transfected by a
plasmid comprising one or more nucleotide sequences as described
herein. The cell can also be infected with a virus or virus-like
particle comprising the nucleic acid molecule. In some embodiments,
the cell is a TIL or a MIL. In some embodiments, the MIL is an
activated MIL. MILs can be activated, for example, by incubating
them with anti-CD3/anti-CD28 beads and appropriate cytokines, e.g.,
under hypoxic conditions. An example of growing the MILs under
hypoxic conditions can found, for example, in WO2016037054, which
is hereby incorporated by reference in its entirety. In some
embodiments, the nucleic acid molecule is transfected into a cell
after the cell has been incubated in a hypoxic environment as
described herein. In some embodiments, the nucleic acid molecule is
transfected into a cell after the cell has been incubated in a
hypoxic environment for about 1, 2, 3, 4, or 5 days. In some
embodiments, the cell is then incubated under normoxic conditions
for about 1, 2, 3, 4, or 5 days.
[0040] In some embodiments, a MIL comprising the chimeric
transmembrane protein is prepared according to a method described
in WO2016037054, which is hereby incorporated by reference in its
entirety. In some embodiments, the method may comprise removing
cells in the bone marrow, lymphocytes, and/or marrow infiltrating
lymphocytes ("MILs") from the subject; incubating the cells in a
hypoxic environment, thereby producing activated MILs; and
administering the activated MILs to the subject. The cells can also
be activated in the presence of anti-CD3/anti-CD28 antibodies and
cytokines as described herein. A nucleic acid molecule encoding a
chimeric transmembrane protein, such as one of those described
herein, can be transfected or infected into a cell before or after
the MIL is incubated in a hypoxic environment.
[0041] The hypoxic environment may comprise less than about 21%
oxygen, such as less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or less than about 3%
oxygen. For example, the hypoxic environment may comprise about 0%
oxygen to about 20% oxygen, such as about 0% oxygen to about 19%
oxygen, about 0% oxygen to about 18% oxygen, about 0% oxygen to
about 17% oxygen, about 0% oxygen to about 16% oxygen, about 0%
oxygen to about 15% oxygen, about 0% oxygen to about 14% oxygen,
about 0% oxygen to about 13% oxygen, about 0% oxygen to about 12%
oxygen, about 0% oxygen to about 11% oxygen, about 0% oxygen to
about 10% oxygen, about 0% oxygen to about 9% oxygen, about 0%
oxygen to about 8% oxygen, about 0% oxygen to about 7% oxygen,
about 0% oxygen to about 6% oxygen, about 0% oxygen to about 5%
oxygen, about 0% oxygen to about 4% oxygen, or about 0% oxygen to
about 3% oxygen. In some embodiments, the hypoxic environment
comprises about 1% to about 7% oxygen. In some embodiments, the
hypoxic environment is about 1% to about 2% oxygen. In some
embodiments, the hypoxic environment is about 0.5% to about 1.5%
oxygen. In some embodiments, the hypoxic environment is about 0.5%
to about 2% oxygen. The hypoxic environment may comprise about 20%,
19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, or about 0% oxygen. In some embodiments, the
hypoxic environment comprises about 7%, 6%, 5%, 4%, 3%, 2%, or 1%
oxygen.
[0042] Incubating MILs in a hypoxic environment may comprise
incubating the MILs, e.g., in tissue culture medium, for at least
about 1 hour, such as at least about 12 hours, 18 hours, 24 hours,
30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 3 days, 4 days, 5
days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13
days, or even at least about 14 days. Incubating may comprise
incubating the MILs for about 1 hour to about 30 days, such as
about 1 day to about 20 days, about 1 day to about 14 days, or
about 1 day to about 12 days. In some embodiments, incubating MILs
in a hypoxic environment comprises incubating the MILs in a hypoxic
environment for about 2 days to about 5 days. The method may
comprise incubating MILs in a hypoxic environment for about 1 day,
2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 day, 9 days, 10
days, 11 days, 12 days, 13 days, or 14 days. In some embodiments,
the method comprises incubating the MILs in a hypoxic environment
for about 3 days. In some embodiments, the method comprises
incubating the MILs in a hypoxic environment for about 2 days to
about 4 days. In some embodiments, the method comprises incubating
the MILs in a hypoxic environment for about 3 days to about 4
days.
[0043] In some embodiments, the method further comprises incubating
the MILs in a normoxic environment, e.g., after incubating the MILs
in a hypoxic environment.
[0044] The normoxic environment may comprise at least about 21%
oxygen. The normoxic environment may comprise about 5% oxygen to
about 30% oxygen, such as about 10% oxygen to about 30% oxygen,
about 15% oxygen to about 25% oxygen, about 18% oxygen to about 24%
oxygen, about 19% oxygen to about 23% oxygen, or about 20% oxygen
to about 22% oxygen. In some embodiments, the normoxic environment
comprises about 21% oxygen.
[0045] Incubating MILs in a normoxic environment may comprise
incubating the MILs, e.g., in tissue culture medium, for at least
about 1 hour, such as at least about 12 hours, 18 hours, 24 hours,
30 hours, 36 hours, 42 hours, 48 hours, 60 hours, 3 days, 4 days, 5
days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13
days, or even at least about 14 days. Incubating may comprise
incubating the MILs for about 1 hour to about 30 days, such as
about 1 day to about 20 days, about 1 day to about 14 days, about 1
day to about 12 days, or about 2 days to about 12 days.
[0046] In some embodiments, the cell is transfected or infected
with a nucleic acid molecule encoding a chimeric transmembrane
protein described herein after being placed in a normoxic
environment or before it is placed in a normoxic environment.
[0047] In some embodiments, the MILs are obtained by extracting a
bone marrow sample from a subject and culturing/incubating the
cells as described herein. In some embodiments, the bone marrow
sample is centrifuged to remove red blood cells. In some
embodiments, the bone marrow sample is not subject to apheresis. In
some embodiments, the bone marrow sample does not comprise
peripheral blood lymphocytes ("PBL") or the bone marrow sample is
substantially free of PBLs. These methods select for cells that are
not the same as what have become to be known as TILs. Thus, a MIL
is not a TIL. TILs can be selected by known methods to one of skill
in the art and can be transfected or infected with the nucleic acid
molecules described herein such that the TILs can express the
chimeric transmembrane protein described herein.
[0048] In some embodiments, the cells are also activated by
culturing with antibodies to CD3 and CD28. This can be performed,
for example by incubating the cells with anti-CD3/anti-CD28 beads
that are commercially available or that can be made by one of skill
in the art. The cells can then be plated in a plate, flask, or bag.
Hypoxic conditions can be achieved by flushing either the hypoxic
chamber or cell culture bag for 3 minutes with a 95% Nitrogen and
5% CO.sub.2 gas mixture. This can lead to, for example, 1-2% or
less O.sub.2 gas in the receptacle. Cells can be then cultured as
described herein or as in the examples of WO2016037054, which is
hereby incorporated by reference.
[0049] In some embodiments, a hypoxic MIL comprising a chimeric
transmembrane protein as described herein is provided. In some
embodiments, the hypoxic MIL is in an environment of about 0.5% to
about 5% oxygen gas. In some embodiments, the hypoxic MIL is in an
environment of about 1% to about 2% oxygen gas. In some
embodiments, the hypoxic MIL is in an environment of about 1% to
about 3% oxygen gas. In some embodiments, the hypoxic MIL is in an
environment of about 1% to about 4% oxygen gas. A hypoxic MIL is a
MIL that has been incubated in a hypoxic environment, such as those
described herein, for a period of time, such as those described
herein. Without being bound to any particular theory, a hypoxic MIL
will undergo changes in protein and/or gene expression that affect
the anti-tumor capabilities of the MIL. As described herein, the
hypoxic MIL can also be activated with the presence of
anti-CD3/anti-CD28 beads or other similar activating reagents.
Thus, a hypoxic MIL can also be an activated-hypoxic MIL.
[0050] In some aspects, the embodiments relates to a method for
increasing an immune response in a subject, comprising
administering to the subject a recombinant cell as described
herein. In some embodiments, the embodiments relate to a method for
treating a neoplasm in a subject, comprising administering to the
subject a recombinant cell as described herein. The neoplasm may be
a benign neoplasm, a malignant neoplasm, or a secondary neoplasm.
The neoplasm may be cancer. The neoplasm may be a lymphoma or a
leukemia, such as chronic lymphocytic leukemia ("CLL") or acute
lymphoblastic leukemia ("ALL"). The neoplasm may be multiple
myeloma as well as any solid tumor (e.g., breast cancer, prostate
cancer, lung cancer, esophageal cancer, brain cancer, kidney
cancer, bladder cancer, pancreatic cancer, osteosarcoma, and the
like).
[0051] The method may comprise administering to the subject a
plurality of recombinant cells as described herein. The method may
comprise administering to the subject an effective amount of
recombinant cells as described herein.
[0052] In some embodiments, the cell is obtained from the subject.
The cell that is transfected or infected may be obtained from the
subject. The cell can be obtained as described herein. For example,
a cell that is administered may be autologous to the subject. In
some embodiments, the cell that is administered is allogeneic to
the subject. The cell may be obtained from the subject and
transfected or infected with a nucleic acid encoding a chimeric
transmembrane protein as described herein. The cell may be a
daughter cell, wherein a parent of the daughter cell was obtained
from the subject. The recombinant cell may have been transfected or
infected with the nucleic acid or a parent of the recombinant cell
may have been transfected or infected with the nucleic acid. In
some embodiments, the cell after being transfected or infected
expresses a protein comprising one or more of the amino sequences
described herein.
[0053] The method may further comprise making the recombinant cell,
wherein making the recombinant cell comprises transfecting or
infecting a cell with a nucleic acid encoding a chimeric
transmembrane protein, such as those described herein. In some
embodiments, the chimeric transmembrane protein comprises an amino
acid sequence set forth in any one of SEQ ID NO: 5, 6, 7, 8, 9, 10,
or 11 or a variant thereof. Similarly, the method may further
comprise making a plurality of recombinant cells, wherein making
the plurality of recombinant cells comprises transfecting or
infecting a plurality of cells with nucleic acids encoding a
chimeric transmembrane protein, such as those described herein. The
method may further comprise expanding a parent cell, e.g., the
recombinant cell may be a daughter cell of the parent cell. The
method may comprise expanding a population of cells, e.g., the
method may comprise administering to the subject a plurality of
recombinant cells as described herein, and each cell of the
plurality of recombinant cells may be a daughter cell of a parent
cell.
[0054] The method may further comprise isolating the cell or a
parent cell from the subject.
[0055] The method may further comprise sorting the cell, e.g., by
fluorescence activated cell sorting ("FACS") or magnetic activated
cell sorting ("MACS").
[0056] The cells can be administered to a subject by any suitable
route in, for example, a pharmaceutically acceptable composition.
In some embodiments, the composition is pyrogen free. For example,
administration of the cells may be carried out using any method
known in the art. For example, administration may be parenteral,
intravenous, intra-arterial, subcutaneous, intramuscular,
intracranial, intraorbital, ophthalmic, intraventricular,
intracapsular, intraspinal, intracisternal, intraperitoneal,
intracerebroventricular, or intrathecal. For parenteral
administration, the cells may be administered by either
intravenous, subcutaneous, or intramuscular injection, in
compositions with pharmaceutically acceptable vehicles or carriers.
The cells can be formulated for parenteral administration by
injection, for example, by bolus injection or continuous infusion.
The compositions can take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and can contain formulatory
agents, for example, suspending, stabilizing, and/or dispersing
agents.
[0057] For administration by injection, it can be desired to use
the cells in solution in a sterile aqueous vehicle which may also
contain other solutes such as buffers or preservatives as well as
sufficient quantities of pharmaceutically acceptable salts or of
glucose to make the solution isotonic. In some embodiments, the
pharmaceutical compositions may be formulated with a
pharmaceutically acceptable carrier to provide sterile solutions or
suspensions for injectable administration. In particular,
injectables can be prepared in conventional forms, either as liquid
solutions or suspensions or as emulsions. Suitable excipients are,
for example, water, saline, dextrose, mannitol, lactose, lecithin,
albumin, sodium glutamate, cysteine hydrochloride, or the like. In
addition, if desired, the injectable pharmaceutical compositions
may contain minor amounts of nontoxic auxiliary substances, such as
wetting agents, pH buffering agents, and the like. Suitable
pharmaceutical carriers are described in "Remington's
pharmaceutical Sciences" by E. W. Martin.
[0058] The subject may be any organism that comprises immune cells.
For example, the subject may be selected from rodents, canines,
felines, porcines, ovines, bovines, equines, and primates. The
subject may be a mouse or a human.
[0059] The subject may have a neoplasm. The neoplasm may be a
benign neoplasm, a malignant neoplasm, or a secondary neoplasm. The
neoplasm may be cancer. The neoplasm may be a lymphoma or a
leukemia, such as chronic lymphocytic leukemia ("CLL") or acute
lymphoblastic leukemia ("ALL"). The subject may have a
glioblastoma, medulloblastoma, breast cancer, head and neck cancer,
kidney cancer, ovarian cancer, Kaposi's sarcoma, acute myelogenous
leukemia, and B-lineage malignancies. The subject may have multiple
myeloma.
[0060] In some embodiments, the subject is a subject "in need
thereof" As used herein, the phrase "in need thereof" means that
the subject has been identified or suspected as having a need for
the particular method or treatment. In some embodiments, the
identification can be by any means of diagnosis. In any of the
methods and treatments described herein, the subject can be in need
thereof.
[0061] As used herein, terms such as "a," "an," and "the" include
singular and plural referents unless the context clearly demands
otherwise.
[0062] As used in this document, terms "comprise," "have," "has,"
and "include" and their conjugates, as used herein, mean "including
but not limited to." While various compositions, and methods are
described in terms of "comprising" various components or steps
(interpreted as meaning "including, but not limited to"), the
compositions, methods, and devices can also "consist essentially
of" or "consist of" the various components and steps, and such
terminology should be interpreted as defining essentially
closed-member groups.
[0063] As used herein, the terms "treat," "treated," or "treating"
mean both therapeutic treatment wherein the object is to slow down
(lessen) an undesired physiological condition, disorder or disease,
or obtain beneficial or desired clinical results. For purposes of
the embodiments described herein, beneficial or desired clinical
results include, but are not limited to, alleviation of symptoms;
diminishment of extent of condition, disorder or disease;
stabilized (i.e., not worsening) state of condition, disorder or
disease; delay in onset or slowing of condition, disorder or
disease progression; amelioration of the condition, disorder or
disease state or remission (whether partial or total), whether
detectable or undetectable; an amelioration of at least one
measurable physical parameter, not necessarily discernible by the
patient; or enhancement or improvement of condition, disorder or
disease. Thus, "treatment of cancer" or "treating cancer" means an
activity that alleviates or ameliorates any of the primary
phenomena or secondary symptoms associated with the cancer or any
other condition described herein. In some embodiments, the cancer
that is being treated is one of the cancers recited herein.
EXAMPLES
[0064] The following examples are illustrative, but not limiting,
of the methods and compositions described herein. Other suitable
modifications and adaptations of the variety of conditions and
parameters normally encountered in therapy and that are obvious to
those skilled in the art are within the spirit and scope of the
embodiments.
Example 1: CAR Transduction Protocol
[0065] 16-24 hours prior to transduction, T-cells were plated in an
appropriate media and were stimulated with CD3, CD28 and IL-2. The
cells were then placed in an incubator (37.degree. C./5% CO.sub.2)
overnight. After 16-24 hours, as much media as possible was removed
without disturbing the cells. The CAR virus was then added to the
cells and placed back in the incubator for 4-12 hours. After 4-12
hours, the appropriate volume of media containing IL-2 was added
back to the cells and then placed back in the incubator. Cells were
left in the incubator to grow, splitting and changing media when
necessary, for 3-12 days. CAR transduction may be checked by a
variety of methods including, but not limited to flow cytometry,
western blotting or fluorescence microscopy, if a fluorescent
reporter gene has been used.
Example 2: CAR Transfection Protocol
[0066] 293T cells were passaged every two days in DMEM+10% FBS for
at least three passages at a cell density at which they never
became more than 80% confluent. One day prior to transfection, the
293T cells were seeded at a density at which they were about 80%
confluent after 24 hours (on the day of transfection). On the day
of transfection, media was removed and enough fresh media was added
to cover the cells. In a separate tube, VSV-G, Gag, Pol & Rev
plasmids, a transfection reagent and the CAR plasmid were combined
and incubated at room temperature for 10-20 minutes. This mixture
was then added drop-wise to the 293T cells and incubated overnight.
12-24 hours after transfection, the media was either completely
changed or additional fresh media was added. At both 48 hrs and 72
hrs post-transfection, virus-containing media from the cells was
collected and cells were replenished with fresh media. Any cells in
the collected media were removed by centrifugation or filtration.
The collected media was then spun in an ultracentrifuge to pellet
the virus. Excess media was removed and the virus was re-suspended
in DMEM or HBSS, aliquoted into sterile tubes and stored at
-80.degree. C. until used.
Example 3: Mil Function and Growth is not Negatively Affected by
the Presence of a Chimeric Receptor Protein
[0067] MILs obtained from subjects were activated and expanded as
described herein. Briefly, after the marrow sample was obtained
from the subject, the cells were incubated under hypoxic conditions
in the presence of anti-CD3/anti-CD28 beads and cytokines as
described in WO2016037054, which is hereby incorporated by
reference. The MILs were then infected with a virus comprising a
nucleic acid molecule encoding a chimeric transmembrane protein
comprising SEQ ID NO: 11. The cells were then grown under normoxic
conditions and allowed to expand. The control and infected MILs
were contacted with different cell types. Neither the expansion of
the MILS nor the ability of the MILs to recognize antigens was
negatively affected by the presence of the chimeric transmembrane
protein. These results demonstrate that adding a chimeric
transmembrane protein to a MIL is not detrimental to its functions
and growth. The results are illustrated in FIG. 6, Panel A and B,
which are from two different patients.
[0068] In summary, the embodiments and examples provided herein
demonstrate that cells expressing a chimeric transmembrane protein
can be effectively used to treat cancer and/or modulate an immune
response.
[0069] Any U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications
and non-patent publications, including CAS numbers, referred to in
this specification and/or listed in the Application Data Sheet are
incorporated herein by reference, in their entirety.
* * * * *